Proteins and nucleic acids employ only a small fraction of the available functionality of these types of molecules. There is currently considerable interest in modifying proteins and nucleic acids to diversify their functionality. Molecular evolution efforts include in vitro diversification of a starting molecule into related variants from which desired molecules are chosen. Methods used to generate diversity in nucleic acid and protein libraries include whole genome mutagenesis (Hart et al., Amer. Chem. Soc. (1999), 121:9887-9888), random cassette mutagenesis (Reidhaar-Olson et al., Meth. Enzymol. (1991), 208:564-86), error-prone PCR (Caldwell et al., PCR Methods Applic. (1992), 2: 28-33) and DNA shuffling using homologous recombination (Stemmer, Nature (1994), 370:389-391). After diversification, molecules with novel or enhanced properties can be selected.
Conventional directed evolution involves discrete cycles of mutagenesis, transformation or in vitro expression, screening or selection, and gene harvesting and manipulation.1,2 In contrast, evolution in nature occurs in a continuous, asynchronous format in which mutation, selection, and replication occur simultaneously. Although successful evolution is strongly dependent on the total number of rounds performed, the labor- and time-intensive nature of discrete directed evolution cycles limit many laboratory evolution efforts to a modest number of rounds.
In contrast, continuous directed evolution has the potential to dramatically enhance the effectiveness of directed evolution efforts by enabling an enormous number of rounds of evolution to take place in a single experiment with minimal researcher time or effort. While laboratories have explored various aspects of continuous evolution, no generalizable, continuous directed evolution system has been reported. In a landmark experiment, Joyce and co-workers engineered a ribozyme self-replication cycle in vitro and used this cycle to continuously evolve a ribozyme with RNA ligase activity (Wright, M. C. & Joyce, G. F. (1997) Science 276, 614-617). However, the foregoing example of continuous directed evolution cannot be easily adapted to evolve other biomolecules.
Continuous directed evolution minimally requires (i) continuous mutagenesis of the gene(s) of interest, and (ii) continuous selective replication of genes encoding molecules with a desired (on-target) activity. Several groups have developed methods to achieve continuous or rapid non-continuous cycles of mutagenesis. For example, Church and coworkers recently developed multiplex automated genome engineering (MAGE), a system capable of generating targeted diversity in E. coli through automated cycles of transformation and recombination (Wang, H. H. et al. (2009) Nature 460, 894-898). While these advances are capable of very efficiently creating gene libraries, they have not been linked to a rapid and general continuous selection and consequently have not enabled continuous directed evolution.